Poultry feed containing mycelium of streptomyces mediolani

ABSTRACT

THIS INVENTION RELATES TO MYCELLIUM OF STREPTOMYCES MEDIOLANI WHICH ARE USEFUL AS AN ADDITIVE TO POULTRY FEED FOR PIGMENT-INCREASING PURPOSES.

nited States Patent 3,822,352 POULTRY FEED CONTAINING MYCELIUM F STREPTOMYCES MEDIOLANI Giovanni Franceschi and Arpad Grein, Milan, Italy, assignors to Societa Farmaceutici Italia, Milan, Italy No Drawing. Original application Nov. 14, 1968, Ser. No. 775,949, now Patent No. 3,598,702, dated Aug. 10, 1971. Divided and this application Aug. 6, 1970, Ser. No. 61,863

Claims priority, application Italy, May 14, 1968, 16,443/ 68 Int. Cl. A61k 27/00 US. Cl. 424-195 1 Claim ABSTRACT OF THE DISCLOSURE This invention relates to mycelium of Streptomyces mediolani which are useful as an additive to poultry feed for pigment-increasing purposes.

This application is a division of our application Ser. No. 775,949 filed Nov. 14, 1968, now US. Pat. No. 3,598,702.

The invention relates to a new process for the preparation of products having a pigmenting action. More particularly, the present invention relates to a microbiological process for the preparation of isorenieratenes of the class consisting of isorenieratene, 3-oxy-isorenieratene and 3,3- dioxy-isorenieratene by means of the new microorganism Streptomyces mediolani.

Isorenieratene is a known product, described in the literature by Yamaguchi, Bull. Chem. Soc. Japan, 1957, 30, p. 111. 3-oxy-isorenieratene and 3,3'-dioxy-isorenieratene are described in Belgian Pat. 687,906. The above products, particularly 3-oxyand 3,3-dioxy-isorenieratene, have a high pigmenting action and are therefore employed in zootechny. These products are prepared according to the literature, by rather complicated chemical syntheses.

We have surprisingly found a microbiological process for the preparation of such compounds which, besides giving good yields, is much simpler and economically more advantageous than the known chemical syntheses. In fact, for the use of the products thus obtained in zootechny, it is not necessary to carry out a separation and purification of the products, but the mycelium in toto containing the pigmenting products may be directly employed as an additive to the commonly used animal diets.

Streptomyces mediolani, designated as strain 2215/74 Fl. of the Farmitalia strain collection, has been deposited at the Institute of Plant Pathology of the University of Milan where it received the number I.P.V. 1952, and at the Institute of Microbiology of Rutgers University, where 3,822,352 Patented July 2, 1974 it received the number I.M.R.U 3934. The strain is freely available from the Institute of Plant Pathology.

The microorganism has the following microscopic and biochemical morphological characteristics.

MICROSCOPIC ASPECT MACROSCOPIC ASPECT Table 1 gives the cultural properties observed on the indicated media, in which the microorganism is grown at 28 C. Observations were made at the 3rd, 8th, 15th and 20th day after inoculation. The microorganism shows substantially a rapid and abundant growth with the formation of a compact, solid vegetative mycelium as a smooth patina, from yolk yellow to orange yellow colored. The aerial mycelium is also abundant on all the cultural media, with a rather cottony aspect, from vanilla yellow to pink yellow or beige yellow colored. No remarkable differences are noted in the aspect of the microorganism grown on synthetic or organic media.

BIOCHEMICAL PROPERTIES Proteolysis of gelatine: positive.

Reduction of nitrates to nitrites: positive. Hydrolysis of starch: positive.

Milk is not coagulated, but peptonized.

Production of melamine: negative.

Decomposition of tyrosine: positive.

Production of hydrogen sulfide: positive.

The microorganism produces no soluble pigments.

The following carbon sources are utilized: glucose, 1- arabinose, saccarose, d-xylose, d-mannitol, d-fructose, maltose, rhaminose and glycerine; however, it does not utilize meso-inositol and rafiinose.

The microorganism does not grow at 50 C. and does not produce sclerotia.

In liquid submerged and shaken culture it produces isorenieratene, 3-oxy-isorenieratene and 3,3dioxy-isorenieratene.

TABLE 1 Soluble Medium Growth Aerial mycelium Vegetative mycelium pigment Bennet's agar Abundant, in smooth Abundant, slightly cottony, vanilla Yolk yellow or apricot yellow; Abs nt.

patina. yellow with beige or rose shades. same backside. Czapecks agar Moderate, in smooth Abundant, eottony; vanilla yellow Lemon yellow to apricot yellow; Do.

patina. with beige-gray shades. same backside. Asparagine-glucose agar Abundant, in smooth Moderate, smooth vanilla yellow... Lemon yellow; same backside..." Do.

patina. Glycerine-glycine agar Abundant, in slightly Abundant, cottony; from rose yellow Apricot yellow; same backside Do.

folded patina. to clearly beige. Emersons agar 1 Abuirdant, in creasy Scanty, smooth, white-cream Orange yellow same backside. Do.

pa ma. Starch agar and salts a Abundant, in smooth Abundant. cottony; from pink do Do.

patina. yellow to light beige. Potato agar 4 -.do Abundant, from dusty to cottony; d0 Do.

pink yellow with light beige shad TABLE 1Continued Soluble Medium Growth Aerial mycelium Vegetative mycelium pigment Oats agar L; d Abundant, rather smooth, dusty; Light orange yellow; same back- Do.

powder rose-yellow. si e. Asparagine-glycerlne agar do Abundant, eottony; from powder Orange yellow; same baekside..... Do.

pink to light beige. Glucose-yeast extract agarh. do Abundant, slightly cottony; from Deep orange yellow; same back- Do.

vanilla yellow to light beige. side. Stareh-peptone agar .do .1--. Alfiufidint, cottony; from yellow to Orange yellow; same baekside Do.

g eige. Potassium nitrate-peptone agar do Moderate, rosy; vanilla yellow with Light orange yellow; same back- Do.

powder pink shades. side.

1 Waksman, S. 11., "The Actinomycetes, Vol. II (1961), pp. 328-334. 2 Pridham, T. G., et al., Antibiotics Annual (1955-1957), pp. 947-953. B Baldacei, E., et al., Giorn. Microbiol. 9, p. 39 (1961).

4 Grain, A., et a1, Giorn. Microbiol. 18, p. 299 (1965).

IDENTIFICATION OF THE MICROORGANISM The properties shown by the microorganism under examination and previously described allow referring it to the genus Streptomyces Waksman et Henrici (Bergeys Manual of Determinative Bacteriology, 1957, pp. 774- 775). The microorganism belongs to the Section Rectusfiexibilis, series yellow of Pridham et al. (Appl. Microbiol. 6, 1958, p. 52).

The colors of the vegetative mycelium and of the aerial mycelium do not allow referring it to any of the most famous subgeneric groups proposed by Waksman (The Actinomycetes, Vol. II, p. 117, 1961), by Baldacci (Glorn. Microbiol. 6, 1958, p. by Gause et al. (Zur Klassifizienlng der Actinomyceten, 1958, p. 17), by Flaig et al. (Arch. Mikrobiol. 35, 105, 1960) and by Ettlinger et al. (Arch. Mikrobiol. 31, 326, 1958). A comparison between the properties of the microorganism under examination and those reported in the literature for the species belonging to the series Yellow" of Pridham et al., has shown that none of the latter has all the properties corresponding to those of the microorganism under examination.

Thus, it is concluded that the microorganism under examination cannot be identified with any of the species described in the literature and is therefore considered a new species and has been called Streptomyces mediolani.

Streptomyces mediolani may be stored by successive inoculations on solid media or by lyophlization of a suspension of its spores in milk. The process according to the invention consists in culturing the new microorganism Streptomyces mediolani, in submerged culture, in a cultural medium containing a carbon and nitrogen source and mineral salts. More particularly, the microorganism is cultured on a liquid cultural medium under aerobic conditions at a temperature between 24 C. and 37 C., preferably at 27 C., over a period of from 72 to 160 hours. The pH may vary according to the media employed, from 6 to 8.

Glucose, dextrin, starch, various meals (maize meal, soya meal, wheat meal, etc.), corn steep liquor and other substances usually employed can be utilized as the carbon source. The nitrogen source, besides the above-mentioned complex substances containing nitrogen, may be casein, cotton seed meal and ammonium salts, such as ammonium sulphate, phosphate, chloride and other substances commonly used. The mineral salts useful for the production of the carotenoids vary according to the medium employed. For example, sodium, potassium, magnesium, iron, zinc, copper and cobalt, chlorides, sulphates or phosphates, calcium carbonate and others commonly used can be employed.

The fermentation may be carried out in rflasks or in laboratory or industrial fermenters of varying capacity. When the fermentation is over, the mycelium is separated from the culture broth by filtration or centrifugation and the pigmenting products contained are isolated by extraction with a suitable organic solvent miscible or immiscible in water such as methanol, chloroform, acetone or methylene chloride. The separation of each product is carried out by chromatography according to known techniques.

The products obtained by the process of the invention can be employed in zootechny and their high pigmenting power has been shown particularly useful when they are given to poultry. The products are added as such or in mixture with feeds or, preferably, the mycelium obtained from the fermentative process can be directly employed. In the first case, the pigmenting product is mixed with one of the ingredients of the diet commonly used for feeding. If the mycelium, separated from the culture broth, is utilized, it is dried with hot air or in vacuum and then milled. The powder thus obtained is deeply mixed with the foodstuif commonly employed. The quantity of pigmenting product which is added to foodstuffs may vary according to the desired pigmentation and according to the optional presence of natural pigmenting elements. Preferably it may be from 0.3 to 6 g. per quintal of foodstuff.

The pigmenting action of the products obtained by the process of the invention has been determined by the color intensity of the yolks measured according to the colorimetric scale Roche (see Mainguy P. et a1. La couleur vitelline Ed. Hoifmann La Roche).

The test had been carried out on 30 laying hens, 7 months old, each being in a single cage and divided in three groups of 10. The treatment was carried out according to the following scheme:

Group Treatment 1 Basie diet containing a small quantity of total pigments corresponding to 13.89 mg./kg.

2 Basic diet plus dehydrated lucerne corresponding to a content of total pigments of 1,500 mg./q. of mixture.

3 Basic diet; plus mycelium corresponding to a content of total pigments of 1,500 mg./l. of mixture.

The composition of the basic diet was as follows: (The values indicate the percentages by weight.)

Farcomplex is a mixture of vitamins and oligoelements having the following composition (the values refer to 1 kg. of the same mixture):

In groups 2 and 3, the dehydrated lucerne and the mycelium have substituted similar quantities of wheat bran. The test lasted 30 days and has been divided in two periods:

('1) preexperimental period 010 days) wherein all the subjects have been fed ad libitum with the basic diet containing no lucerne;

(2) experimental period 1019 days) wherein the subjects have been fed as above described.

During the experimental period and on alternate days the pigmentation of the yolk has been measured. The [following Table 2 reports the minimum, average and maximum results.

TABLE 2 Experimental periodRoehe scale Group Minimum Average Maximum The following examples illustrate the invention without limiting it.

EXAMPLE 1 The sporulated surface of a .10 day old culture of Streptomyces mediolani, grown in a test tube on agarpotato, glucosate at 2'8" C., was removed and collected in 4 ml. of sterilized distilled water. 0.5 ml. of this suspension was used to inoculate a 300 ml. flask containing 60 ml. of the following medium:

'Percent Dextrine 4 Casein 1 Calcium carbonate 0.5 Ammonium sulphate 0.1 Bipotassium phosphate 0.0 Corn steep liquor '1 Tap water to 100 ml.

EXAMPLE 2 The operation was as in Example 1 with the difference that the medium employed for the productive phase had the following composition:

Percent Insoluble starch 4.5 Soya meal 2.25 Corn steep liquor 2.35 Sodium chloride 0.5

Calcium carbonate 0.35 Tap water to ml.

After hours of fermentation, a production of 600v of pigmenting products per ml. of culture broth was obtained.

EXAMPLE 3 The operation was as in Example 1 with the diiference that the medium employed for the productive phase had the following composition:

Percent Glucose 0.3 Cotton seed meal 0.4 Soluble starch 7 Calcium carbonate 1.2 Ammonium sulphate 0.75 Corn steep liquor 2.35

Tap water to 100 ml.

After 120 hours of fermentation, a production of 900 of pigmenting products per m1. of culture broth was obtained.

EXAMPLE 4 The operation was as in Example 1 with the difference that the medium employed for the productive phase has Magnesium sulphate 0.1 Tap water to 100 ml.

After 69 hours of fermentation, a production of 700y of pigmenting products per ml. of culture broth was obtained.

EXAMPLE 5 Three 300 ml. flasks containing 60 ml. of the medium described in Example 1 were inoculated with a 20 day old culture of Streptomyces mediolani, grown on the medium and under the conditions described in Example 1. The flasks were incubated at 28 C. for 26 hours under the conditions described in Example 1.

ml. of the culture broth thus obtained were inoculated in 300 ml. of the same liquid medium, contained in a 5 l. fermenter provided with a screw-stirrer, an inlet tube for bubbling in air ending under the screw-stirrer, a breakwater device, tubes for inoculation, air outlet tubes and temperature checking equipment. The operation was carried out at 28 C. with an aeration rate of 3 liters per minute and under stirring with a rate of 400 r.p.m. During fermentation, foaming was checked by adding small quantities of silicone antifoaming agent. The maximum yield of pigmenting products was obtained after 77 hours of fermentation. This was 500 per ml. of culture broth.

10 liters of culture broth were filtered with 2% of Hyflo Supercel (Registered Trademark), the filtrate was eliminated. The mycelium was suspended at room temperature in 3 l. of methanol and shaken. Three extractions were then carried out with 3 liters of a mixture of methanolchloroform (1:1) each time. The combined extracts were repeatedly washed with water and the chloroform layer separated was dried over sodium sulphate and evaporated in vacuo at 35 C. The dark-red oily residue consists of a pigment complex. This residue was taken up with petroleum ether (300 cc.), and a brown-red solid separated by filtration weighs 1.5 g. The ethereal mother liquors were concentrated to about 100 ml. and were chromatographed over a column of neutral alumina, first by elution with petroleum ether to eliminate fats, then with petroleum ether containing 1% of acetone. The eluate of the large top band was concentrated to small volume and allowed to stand overnight in a refrigerator. 400 mg. of purplered crystalline product melting at 200-201 C. were separated. Adsorption maxima in the visible spectrum were at the following wavelengths:

(FRACTION A) Solvent: Amax. (m Petroleum ether (428), 448, 478 Benzene (440), 465, 495

By chromatography on a thin layer of silica gel, it has been found: R =O.85 (benzene) and R =0.38 (petroleum ether and 1% of acetone). This fraction A has been therefore identified as isorenieratene by chromatographic and spectrophotometric comparison and by melting point in mixture with a sample prepared by synthesis.

Continuing the elution of the column with petroleum ether containing 10% of acetone, a second band was collected. From the eluate concentrated to small volume, 200 mg. of a brick-red powder were obtained. This powder melting at 180185 C. has adsorption maxima in the visible spectrum at the following wave lengths:

(FRACTION B) Solvent: kmax. (my) Petroleum ether (425), 446, 475 Benzene (438), 463, 493

By chromatography on a thin layer of silica gel was obtained: R;=0.47 (benzene) and R ==0.7 (methylene chloride).

Fraction B is slightly soluble in the common organic solvents, while it is very soluble in alcoholic sodium carbonate with a bright red coloring and it was reprecipitated by acidification. By treatment with acetic anhydride in pyridine it gives a phenolic acetate which is insoluble in alcoholic sodium carbonate.

(FRACTION C) Solvent: 7imax. (mp) Petroleum ether (425), 446, 475 Benzene (438), 463, 493

By chromatography on a thin layer of silica gel there was obtained: R =O.1 (benzene) and R =0.26 (methylene chloride).

This fraction C has been identified with the 3,3'-dioxyisorenieratene by chromatographic and spectrophotometric comparison and by mixed melting point.

We claim:

1. A poultry feed comprising a major portion of a basic feed and an elfective pigment-increasing amount of the dried and milled mycelium of the Streptomyces mediolani.

References Cited UNITED STATES PATENTS 2,974,044 3/1961 Farrow et al 99-4 3,330,737 7/1967 Marnati et al. 999

FOREIGN PATENTS 687,906 2/ 1966 Belgium 99-4 ALBERT T. MEYERS, Primary Examiner F. E. WADDELL, Assistant Examiner U.S. Cl. X.R. 424356 ft P0-1050 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION l'6 -9--DI'V Patent No. 3,822,352 Date-d .lu-lv 2 1974 lnve'n'to'fls), GIOVANNI FRANCESCHI and ARlAD GREIN It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

F I v I g Column 1, line 10: "16,443/68 should read 16,443 A/68 Column 2, line 44: "saccarose" should read saccharose Column 3, line 29: "(G1orn. should read (Giorn.

Column 4, line 54: "totalpigments of 1,500 mg./l. should read total pigments of 1,500 mg./q.

Column 6, line 56: "300 ml." should read 3,000 ml.

Signed and sealed this 15th day of October 1974.

(SEAL) Attest:

MCCOY M. GIBSON. JR. C. MARSHALL DANN Attesting Officer Commissioner of Patents 

